Serine protease inhibitors or "serpins" are a superfamily of inhibitors involved in the mediation of a variety of biological processes essential to survival of a host. Members of the serpin family play a role in a great number of biological processes including, but not limited to, inflammation, fertilization, tumor migration, neurotropism, and heat shock. Maspin was recently identified and characterized as a protective serpin normally present in mammary epithelium but absent from most mammary carcinoma cell lines. Serpins are found in plants, prokaryotes, insects and animals. Natural mutations and modifications of serpins are correlated with a number of serious disease states. Serpin dysfunction is associated with lung, liver and blood coagulation diseases such as emphysema, liver cirrhosis, thrombosis and pulmonary embolism.
The interaction of serpins with endogenous and microbial proteases produces a spectrum of molecular species, each of which are components of a highly evolutionarily conserved homeostatic mechanism that operates to maintain concentrations of intact, active serpins essential to a host's survival. For example, the serpin-protease complex and the hydrolyzed, inactive form of the intact serpin stimulate the production of interleukin-6, signaling hepatocytes to increase synthesis of the acute phase proteins including a subpopulation of the serpin superfamily of proteins. While serpin-enzyme complexes are rapidly cleared from the circulation, cleaved and intact forms of these complexes can accumulate in local areas of inflammation. This accumulation establishes a complex microenvironment of chemoattractants and inhibitors of chemotaxis as well as activators and inhibitors of neutrophil degranulation, leukotrienes, platelet activating factor (PAF), and superoxide production. The extreme virulence of several pox viruses has been attributed in part to a serpin whose target is cysteine proteinase ICE, the interleukin 1-.beta. converting enzyme.
Through various animal models, it has been demonstrated that uncontrolled serine protease activity is a major mechanism of lung injury and that an appropriate serpin response controls the degree of the injury. For example, antithrombin III (ATIII) in combination with .alpha.-1-protease inhibitor (.alpha.1P1), protected sheep from endotoxin-induced lung injury where the individual serpins were not as effective as the combination. Redens et al., Circ. Shock 1988, 26, 15. Redens et al. also showed that ATIII protects against the development of disseminated intravascular coagulation in endotoxemic rats. Emerson et al., Circ. Shock 1987, 21, 1. A scavenger of H.sub.2 O.sub.2 and a chloromethyl ketone inhibitor of elastase blocked reactive oxygen potentiation of neutrophil elastase-mediated acute edematous lung injury in a rat and .alpha.1P1 diminished bleomycin-mediated pulmonary inflammation as well as subsequent fibrosis. Baird et al., Physiol. 1986, 61, 2224 and Nagai et al., Am. Rev. Resp. Dis. 1992, 145, 651. In another system, however, neutrophil elastase inhibitors, Eglin C and a low molecular weight compound L 658,758, failed to inhibit leukotriene B4-induced-neutrophil-mediated adherence, diapedesis or vascular leakage. Rosengren et al., Am J. Physiol. 1990, 259, H1288. As shown by these studies, inhibitors of proteolytic enzymes administered therapeutically can limit the molecular and cellular mechanism of inflammation and reduce tissue damage.
There are two subfamilies within the serpin superfamily. One family contains proteins for which no cognate serine proteases have yet been identified. Examples of proteins in this subfamily include ovalbumin, angiotensinogen and steroid binding globulins. The second family contains members for which at least one serine protease can be found as an inhibitory target. The subfamily of serpins that inhibit serine proteases have characteristic properties that define the activity of the inhibitor, i.e., second order rate constants for inhibition of their cognate enzyme range between 10.sup.2 and 10.sup.7 M.sup.-1 s.sup.-1 ; the enzyme-inhibitor complex is stable under certain conditions and can be detected as a species with a molecular weight greater than the individual components in SDS polyacrylamide gels; and, a large conformational change occurs upon cleavage of the sessile bond in the reactive center leading to increased thermal stability of the protein. An example of a serpin in this subfamily is .alpha.1-antichymotrypsin (ACT), an inhibitor of chymotrypsin (Chtr). ACT is synthesized predominantly by the liver and is one of the acute phase reactants with levels rising rapidly to more than 5 fold in response to a wide variety of injuries including surgery, acute myocardial infarctions, burns, autoimmune diseases, malignancies, infections and liver allograft rejection. ACT has also been linked with the plasticity of the nervous system and associated with beta amyloid deposits in Alzheimer's disease, in aging brain, Down's syndrome and in the Dutch variant of hereditary cerebral hemorrhage with amyloidosis. It has been demonstrated that both native ACT and recombinant ACT (rACT) inhibit superoxide generation by human neutrophils in suspension.
ACT appears to be unique among serpins in its ability to bind to DNA. Katsunama et al., Biochem. Biophys. Res. Commun. 1980, 93, 552. This property is also retained by the nonglycosylated recombinant form, rACT, expressed in E. coli. Rubin et al., J. Biol. Chem. 1990, 265, 1199. ACT, derived from serum, is found in carcinoma cell nuclei (Takada et al., Gann 1982, 73, 742; Takada et al., Cancer Res. 1986, 46, 3688), as well as in the nuclei of non-malignant cells, including those from human neural tissue (Abraham et al., Cell 1988, 52, 487). ACT has been shown to inhibit DNA synthesis in permeabilized human carcinoma cells. Tsuda et al., Biochem. Biophys. Res. Commun. 1987, 144, 409. It has been suggested that this inhibition is a result of ACT inhibition of DNA polymerase alpha (Tsuda et al., Cancer Res. 1986, 46, 6139) and/or of DNA primase (Takada et al., Biochem Int. 1988, 16, 949). ACT also inhibits natural killer cells, which are responsible for tumor lysis, and it has been suggested that ACT present in the nucleus may act as a protective agent for tumor cells. Travis, J. and Salvesen, G. S., Annu. Rev. Biochem. 1983, 52, 655. It is also believed that ACT may modulate the level of chymotrypsin-like enzyme activity found in chromatin. Travis, J. and Salvesen, G. S., Annu. Rev. Biochem. 1983, 52, 655. ACT may also play a role in cell death and apoptosis.
Using site-directed mutagenesis and chemical modification, specific regions of the rACT amino acid sequence have been identified which are important elements for DNA binding of ACT.